This is a competitive renewal which has focused on the role of PI 3-kinase in insulin action. Over the past 4 years, we have defined the similarities, differences and complementary roles of various isoforms of PI 3-kinase regulatory subunits in insulin action. This has been accomplished by creation and characterization of mice and cell lines in which specific isoforms of PI 3-kinase have been deleted or overexpressed. This has led to new hypotheses about the important role of stoichiometry between regulatory and catalytic subunits in insulin action, the potential for other signals emanating from the regulatory subunit of PI 3-kinase involved in regulation of PIP3 half-life and the stress kinase pathways, and the role of Akt as a downstream effector of PI 3-kinase. In the next five-year period, we propose to extend our previous observations and focus on these hypotheses in a series of new specific aims: 1. Define the role of the N-terminal domains of p85alpha/beta and the short isoforms p50alpha and p55alpha/AS53 in insulin signaling, including the potential roles of these domains in signaling mediated by the regulatory subunits independent of PI 3-kinase activity, especially the JNK and p38 MAPK pathways, and the link between p85 and lipid phosphatase PTEN, and their differential ability to generate and stabilize PIP3. 2. Define the specific molecules interacting with the N-terminal region of p85, p55 and p50 regulatory subunits through yeast and bacterial two-hybrid screening, determine their role in signaling and subcellular localization of the PI 3-kinase enzyme, and their relationship to signaling through Racl and cdc42. 3. Explore the role of stoichiometry in regulation of in insulin sensitivity by defining the mechanisms regulating expression of various isoforms of regulatory subunit in normal and pathological states and analyzing the promoters of the p85a, p55a and p50a gene in vivo and in vitro. 4. Determine the role of stoichiometry of PI 3-kinase catalytic subunits p110alpha and p110beta (equivalent to age-1 in C. elegans) in insulin signaling, insulin sensitivity and longevity, and define the role of the major downstream effector Aktl in rodents and cell lines by creating and characterizing tissue specific knockout mice alone and in combination with Akt2 knockout.